Hematopoietic stem cells (HSCs) are rare cells that have been identified in fetal bone marrow, umbilical cord blood, adult bone marrow, and peripheral blood, which are capable of differentiating into each of the myeloerythroid (red blood cells, granulocytes, monocytes), megakaryocyte (platelets) and lymphoid (T-cells, B-cells, and natural killer) cells lineages. In addition these cells are long-lived, and are capable of producing additional stem cells, a process termed self-renewal. Stem cells initially undergo commitment to lineage restricted progenitor cells, which can be assayed by their ability to form colonies in semisolid media. Progenitor cells are restricted in their ability to undergo multi-lineage differentiation and have lost their ability to self-renew. Progenitor cells eventually differentiate and mature into each of the functional elements of the blood.
HSC are used in clinical transplantation protocols to treat a variety of diseases including malignant and non-malignant disorders.
HSCs obtained directly from the patient (autologous HSCs) are used for rescuing the patient from the effects of high doses of chemotherapy or used as a target for gene-therapy vectors. HSCs obtained from another person (allogeneic HSCs) are used to treat haematological malignancies by replacing the malignant haematopoietic system with normal cells. Allogeneic HSCs can be obtained from siblings (matched sibling transplants), parents or unrelated donors (mismatched unrelated donor transplants). About 45,000 patients each year are treated by HSC transplantation. Although most of these cases have involved patients with haematological malignancies, such as lymphoma, myeloma and leukaemia, there is growing interest in using HSC transplantation to treat solid tumours and non-malignant diseases. For example, erythrocyte disorders such as β-thalassaemia and sickle-cell anemia have been successfully treated by transplantation of allogeneic HSCs.
The search for factors that can stimulate HSC self-renewal has proven difficult, but recent reports indicate that selected molecules (sonic hedgehog (Bhardwaj), jagged1 (Karanu, 2000), fibroblast growth factor 1 (de Haan, 2003) and Wnt-3a (Willert, 2003)) can both, support maintenance or induce modest expansion of HSC. However, to date the HOXB4 transcription factor has proven to be the most potent stimulator of HSC self renewal (Antonchuk, 2001; Antonchuk, 2002). Similar effect of retrovirally driven ectopic expression of HOXB4 has also been reported for human cells (Buske, 2002; Schiedlmeier, 2003). In addition, it has been shown that recombinant TAT-HOXB4 protein, when added to the HSC culture, could penetrate the cell membrane and provides significant HSC expansion stimuli (Krosl, 2003; US 2004/0082003) and similar effect of stroma cell derived HOXB4 on human HSC has also been reported (Ansellem, 2003). Human HSC, assessed with NOD/SCID SRC assay, can be efficiently and significantly expanded ex vivo using TAT-HOXB4 protein (Krosl, 2005a; Krosl, 2005b). One of the major advantages of TAT-HOXB4 expansion is the fact that it can be performed using recombinant protein, that is without possible drawbacks of gene transfer protocols (Baum, 2004; Modlich, 2005; Woods, 2006).
The major impediment for the use of HOXB4 in clinical setting with or without a PTD (protein transduction domain) such as that of the N-terminal of TAT is its short intracellular and extracellular half-life (40-60 minutes and 3-4 hours, respectively) (Krosl, 2003). With expansion times lasting 4-8 days, that translates into extensive culture manipulation increasing the risk of culture contamination. More stable HOXB4 molecules with similar HSC expansion capabilities would significantly increase its usability in clinical settings.
DNA binding activity of HOXB4 is required to induce HSCs expansion but not its collaboration with PBX1 (Beslu, 2004), even more PBX1 might be a negative regulator of HOXB4 (Krosl, 2003). More recently, the over expression of associated with the down regulation of PBX1 were shown to be able to sustained in vitro symmetrical (i.e. symmetrical division: production of two identical cells by opposition to asymmetrical division: division into one identical cell and one differentiated cell) self-renewal divisions of HSCs. Modulation of transcription of cell cycle regulators induced by HOXB4 over expression in primitive hematopoietic cells, was more pronounced with inhibition of PBX1 expression in these cells (Cellot, 2007) (see also co-pending US 2006/0121566).
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.